The cell-end protein Tea4 spatially regulates hyphal branch initiation and appressorium remodeling in the blast fungus Magnaporthe oryzae.

The differentiation of specialized infection cells, called appressoria, from polarized germ tubes of the blast fungus Magnaporthe oryzae, requires remarkable remodeling of cell polarity and architecture, yet our understanding of this process remains incomplete. Here we investigate the behavior and role of cell-end marker proteins in appressorium remodeling and hyphal branch emergence. We show that the SH3 domain-containing protein Tea4 is required for the normal formation of an F-actin ring at Tea1-GFP-labeled polarity nodes, which contributes to the remodeling of septin structures and repolarization of the appressorium. Further, we show that Tea1 localizes to a cortical structure during hyphal septation which, unlike contractile septin rings, persists after septum formation, and, in combination with other polarity determinants, likely spatially regulates branch emergence. Genetic loss of Tea4 leads to mislocalization of Tea1 at the hyphal apex and with it, impaired growth directionality. In contrast, Tea1 is largely depleted from septation events in Δtea4 mutants and branching and septation are significantly reduced. Together, our data provide new insight into polarity remodeling during infection-related and vegetative growth by the blast fungus.

Septum-associated microtubule organizing centers within conidia support infectious development by the blast fungus Magnaporthe oryzae.

Cytoplasmic microtubule arrays play important and diverse roles within fungal cells, including serving as molecular highways for motor-driven organelle motility. While the dynamic plus ends of cytoplasmic microtubules are free to explore the cytoplasm through their stochastic growth and shrinkage, their minus ends are nucleated at discrete organizing centers, composed of large multi-subunit protein complexes. The location and composition of these microtubule organizing centers varies depending on genus, cell type, and in some instances cell-cycle stage. Despite their obvious importance, our understanding of the nature, diversity, and regulation of microtubule organizing centers in fungi remains incomplete. Here, using three-color fluorescence microscopy based live-cell imaging, we investigate the organization and dynamic behavior of the microtubule cytoskeleton within infection-related cell types of the filamentous fungus,Magnaporthe oryzae, a highly destructive pathogen of rice and wheat. We provide data to support the idea that cytoplasmic microtubules are nucleated at septa, rather than at nuclear spindle pole bodies, within the three-celled blast conidium, and provide new insight into remodeling of the microtubule cytoskeleton during nuclear division and inheritance. Lastly, we provide a more complete picture of the architecture and subcellular organization of the prototypical blast appressorium, a specialized pressure-generating cell type used to invade host tissue. Taken together, our study provides new insight into microtubule nucleation, organization, and dynamics in specialized and differentiated fungal cell types.

Turgor-dependent and coronin-mediated F-actin dynamics drive septin disc-to-ring remodeling in the blast fungus Magnaporthe oryzae.

The fungus Magnaporthe oryzae uses a specialized pressure-generating infection cell called an appressorium to break into rice leaves and initiate disease. Appressorium functionality is dependent on the formation of a cortical septin ring during its morphogenesis, but precisely how this structure assembles is unclear. Here, we show that F-actin rings are recruited to the circumference of incipient septin disc-like structures in a pressure-dependent manner, and that this is necessary for their contraction and remodeling into rings. We demonstrate that the structural integrity of these incipient septin discs requires both an intact F-actin and microtubule cytoskeleton and provide fundamental new insight into their functional organization within the appressorium. Lastly, using proximity-dependent labeling, we identify the actin modulator coronin as a septin-proximal protein and show that F-actin-mediated septin disc-to-ring remodeling is perturbed in the genetic absence of coronin. Taken together, our findings provide new insight into the dynamic remodeling of infection-specific higher-order septin structures in a globally significant fungal plant pathogen.

Autophagy machinery promotes the chaperone-mediated formation and compartmentalization of protein aggregates during appressorium development by the rice blast fungus.

The chaperone-mediated sequestration of misfolded proteins into specialized quality control compartments represents an important strategy for maintaining protein homeostasis in response to stress. However, precisely how this process is controlled in time and subcellular space and integrated with the cell's protein refolding and degradation pathways remains unclear. We set out to understand how aggregated proteins are managed during infection-related development by a globally devastating plant pathogenic fungus and to determine how impaired protein quality control impacts cellular differentiation and pathogenesis in this system. Here we show that in the absence of Hsp104 disaggregase activity, aggregated proteins are spatially sequestered into quality control compartments within conidia, but not within terminally differentiated infection cells, and thus spatial protein quality control is cell type-dependent. We demonstrate that impaired aggregate resolution results in a short-term developmental penalty but has no significant impact upon appressorium function. Finally, we show that, somewhat unexpectedly, the autophagy machinery is necessary for the normal formation and compartmentalization of protein aggregates. Taken together, our findings provide important new insight into spatial protein quality control during the process of terminal cellular differentiation by a globally important model eukaryote and reveal a new level of interplay between major proteostasis pathways.

Long-distance early endosome motility in Aspergillus fumigatus promotes normal hyphal growth behaviors in controlled microenvironments but is dispensable for virulence.

In filamentous fungi, early endosomes are continuously trafficked to, and from, the growing hyphal tip by microtubule-based motor proteins, serving as platforms for the long-distance transport of diverse cargos including mRNA, signaling molecules, and other organelles which hitchhike on them. While the cellular machinery for early endosome motility in filamentous fungi is fairly well characterized, the broader physiological significance of this process remains less well understood. We set out to determine the importance of long-distance early endosome trafficking in Aspergillus fumigatus, an opportunistic human pathogenic fungus that can cause devastating pulmonary infections in immunocompromised individuals. We first characterized normal early endosome motile behavior in A. fumigatus, then generated a mutant in which early endosome motility is severely perturbed through targeted deletion of the gene encoding for FtsA, one of a complex of proteins that links early endosomes to their motor proteins. Using a microfluidics-based approach we show that contact-induced hyphal branching behaviors are impaired in ΔftsA mutants, but that FtsA-mediated early endosome motility is dispensable for virulence in an invertebrate infection model. Overall, our study provides new insight into early endosome motility in an important human pathogenic fungus.